An integral component of biotechnology research is the application of protein engineering methods to design or modify proteins. In general, these protein purification methods help in optimising protein properties for particular industrial applications.
With these techniques, scientists need to isolate and purify the specified proteins to allow the study of conformations and substrate specificities. Also, needing study are specific enzyme activities and the reactions with other ligands (a protein that adheres to a receptor protein).
The level of protein purity needed relies on the expected end use of the protein. A crude extract is enough for some applications. Other applications, such as in pharmaceuticals and foods, a high degree of protein is needed. A protein purification company applies several methods to obtain a required purity level.
Create a Strategy
Usually, each protein purification step results in some level of product loss. Therefore, the best protein purification strategy should reach the highest degree of purification in the fewest steps.
In general, the choice of which steps to apply relies on the solubility, charge, size, and other properties of the specified protein. When it comes to purifying a single cytosolic protein, the following methods are most appropriate.
It is worth noting that the purifying of cytosolic protein complexes is often more complicated and demands that different techniques be used.
Prepare a Crude Extract
When purifying intracellular (within the cell) proteins, the first step is to prepare a crude extract. In general, the extract will consist of a complex combination of each protein from the cell cytoplasm, and a few additional macromolecules, nutrients, and cofactors.
This crude extract might be applied in several applications in biotechnology. If purity is a problem, however, subsequent purification steps should be followed. Typically, the preparation of crude protein extracts requires the removal of cellular debris created by cell lysis – this is achieved using enzymes, chemicals, sonication, or a French Press.
Eradicate Debris From the Extract
Centrifugation is the process used to remove the debris, and the supernatant (the liquid formed above a solid residue) is retrieved. Crude preparations (out of the cell) proteins might be reached by simply eradicating the cells by centrifugation.
In some biotechnology applications, there is an increasing need for thermostable enzymes- which can withstand extreme temperatures without denaturing, and ensuring high specific activity at the same time.
Intermediate Protein Purification Steps
Typically, modern biotech protocols take advantage of the numerous commercially available kits or techniques that offer ready-made solutions for typical procedures. In most cases, protein purification is carried out using prepared gel-filtration columns and filters.
The dialysis kit contains instructions that you should follow and ensure to add the appropriate volume of the proper solution and wait for the given length of time as you collect the eluant – the solvent passed via the column – in some fresh test tube.
Generally, chromatographic techniques are applied using automated HPLC equipment or bench-top columns. Separation by HPLC can be performed by ion-exchange, reverse-phrase, or size-exclusion techniques, and samples detected by laser technology or diode array.
Immunoblotting is basically a protein visualisation method applied together with affinity chromatography. The antibodies for a target protein are applied as ligands for an affinity chromatography column.
The specific protein usually remains on the column and is then eliminated by rinsing the column using a salt solution or alternative agents. Antibodies associated with dye labels or radioactive help in the detection of the target protein after it is isolated from the rest of the mixture.